Hetero-oligomerization-dependent Binding of Pig Oocyte Zona Pellucida Glycoproteins ZPB and ZPC to Boar Sperm Membrane Vesicles

Abstract

The zona pellucida surrounding the pig oocyte contains two M r 55,000 glycoproteins, pZPB and pZPC, which are orthologues of mouse zona proteins ZP1 and ZP3, respectively. We previously reported that isolated boar sperm membrane vesicles possess high affinity binding sites for partially purified pZPB, but not pZPC. Interestingly, co-incubation experiments also implicated pZPB-pZPC complexes as potential ligands. We now report that when depleted of a minor pZPC contaminant by size exclusion chromatography, pZPB lacks independent binding activity. In solid phase binding assays employing immobilized boar sperm membranes, pZPB failed to compete with biotin-(pZPB؉pZPC) probe, and biotin-labeled pZPB yielded negligible binding. However, when co-incubated with pZPC prior to the binding assays, pZPB acted as a potent competitor, and biotin-labeled pZPB exhibited high affinity, saturable binding. Binding activity was attributed to pZPB-pZPC heterocomplexes, which were detected in co-incubation mixtures by size exclusion chromatography and Western blot analysis. In the pig, therefore, sperm membranes possess a zona-binding protein with high affinity sites for pZPB-pZPC heterocomplexes, but not free glycoprotein subunits. Consequently, associative interactions between zona molecules can contribute toward both the assembly of the zona matrix and generation of ligands important for sperm-zona interactions.The zona pellucida is a morphologically discrete, extracellular matrix which envelops and protects the oocyte and preimplantation embryo. This egg investment also actively participates in the fertilization process. The zona pellucida provides docking sites for species-specific sperm attachment and induces bound sperm to undergo the acrosome reaction, the preparatory event for zona penetration and eventual sperm-egg fusion. This intimate association of male and female gametes is mediated in part by sperm-adhesive glycoproteins within the zona matrix and complimentary zona-binding proteins on the sperm surface. Interestingly, although multiple gamete adhesion molecules have been characterized at the molecular level (1-11), the relative physiological importance of many remains a topic of considerable debate (12).Zonae pellucidae contain a repertoire of glycoproteins encoded by three gene families that are conserved across species. Adopting the terminology of Harris et al. (13), the three cDNA-predicted precursor polypeptides in the pig (10,13,14) are herein designated pZPA (79 kDa), pZPB (59 kDa), and pZPC (46 kDa). Due to post-translational processing, the three mature glycoproteins from isolated zonae appear on nonreducing SDS gels as two diffuse bands (15); pZPA runs at M r 90,000, whereas pZPB and pZPC co-migrate at M r 55,000. Electrophoretic or chromatographic protocols permit isolation of copurified pZPB and pZPC glycoproteins (15-17) and such pZP(BϩC) preparations exhibit several important biological activities, including: stimulation of the acrosome reaction in capacitated boar sperm (18), inhibition of sperm-zona att...

Localization of N‐linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida

“…This site is also cleaved in at least half of the ZPA molecules during preparation of the zona from ovarian eggs. The molecular masses of the N-and C-terminal fragments of the endo-b-Galactosidase-digested ZPA are 21 and 63 kDa, respectively [12], and the time course of the N-glycanase (glycopeptidase F; Takara Shuzo, Kyoto, Japan) digestion of these fragments could be monitored by SDS/PAGE on 15 and 8% acrylamide gels, respectively, under reducing conditions [17]. The digestion was performed in 0.1% SDS, 10 mM o-phenanthroline, 100 mM sodium phosphate (pH 8.6) at 37°C and aliquots were removed at various times between 0 min and 22 h and subjected to SDS/PAGE.…”

“…Moreover, the isolated N-terminal peptide of ZPB including Asn220 has sperm-binding activity [11]. Yurewicz et al [12] showed that ZPB and ZPC form heterocomplexes that have sperm-binding activity, but that monomeric ZPB or ZPC does not exhibit this activity. These results suggest that ZPC contributes to the expression of the sperm-binding activity of the neutral N-linked chain of ZPB.…”

Sperm Proteasome as a Putative Egg Coat Lysin in Mammals

“…There is evidence that the sperm receptor complex ZPB-ZPC on the porcine ZP is ubiquitinated. In porcine oocytes, the ZPB-ZPC complex has been shown to be responsible for sperm-ZP binding (Yurewicz et al 1998 ). Furthermore, the porcine ZPC homologues in mouse (ZP3) and human (ZP3) have been implicated in sperm binding and induction of acrosomal exocytosis (Shur et al 2006 ;Gupta et al 2009 ).…”

“…One that continues to be debated, despite decades of research, concerns the mechanism utilized by the fertilizing spermatozoa to penetrate the oocyte vitelline coat (VC), the zona pellucida (ZP). When a capacitated, fertilization-competent spermatozoon binds to the sperm receptor on the ZP, it undergoes acrosomal exocytosis (AE), which causes vesiculation of the acrosomal membrane and exocytosis of the acrosomal cap (Yurewicz et al 1998 ;Bleil and Wassarman 1980 ). This step exposes the acrosome-borne proteolytic enzymes and results in the formation of the acrosomal shroud, which allows the spermatozoa to create a local microenvironment that supports the opening of the fertilization slit and penetration of the sperm through the ZP (Yurewicz et al 1998 ).…”

“…When a capacitated, fertilization-competent spermatozoon binds to the sperm receptor on the ZP, it undergoes acrosomal exocytosis (AE), which causes vesiculation of the acrosomal membrane and exocytosis of the acrosomal cap (Yurewicz et al 1998 ;Bleil and Wassarman 1980 ). This step exposes the acrosome-borne proteolytic enzymes and results in the formation of the acrosomal shroud, which allows the spermatozoa to create a local microenvironment that supports the opening of the fertilization slit and penetration of the sperm through the ZP (Yurewicz et al 1998 ). There are two schools of thought about the mechanism of sperm-ZP penetration: (1) the mechanical force of the sperm tail motility is suffi cient to fully penetrate the ZP (Green and Purves 1984 ;Bedford 1998 ); and (2) an enzyme originating from the acrosome of the fertilizing spermatozoa acts as a putative zona lysin that digests the fertilization slit in the ZP and allows sperm to penetrate through the ZP (Austin and Bishop 1958 ;Austin 1975).…”